Suicide gene therapy is an attractive approach to treatment of cancer because it is more selective than traditional cancer chemotherapy. We have focused on the herpes simplex virus thymidine kinase (HSV-TK), the initial activator of the antiviral drug ganciclovir (GCV) to its cytotoxic triphosphate, because of the superior cytotoxicity of GCV and its unique mechanism of action. The major limitation of gene therapy is low transfer of the suicide gene to tumor cells, and thus all gene therapy approaches must have a mechanism for killing non-transgene-expressing (bystander) cells. HSV-TK/GCV relies on gap junctional intercellular communication (GJIC) to transfer the cytotoxic triphosphate from HSV-TK-expressing to bystander cells. In the previous funding period, we evaluated pharmacologic modulation, based on the mechanism of action for GCV, vs. increased GJIC to enhance therapy with HSV-TK/GCV. The results demonstrated that pharmacologic modulation (with ribonucleotide reductase inhibitors dFdCyd or hydroxyurea) was more efficacious than enhancing GJIC. Furthermore, in a nude mouse model with human tumor xenografts in which only 10% to 50% of the cells expressed HSV-TK, we demonstrated that neither GCV nor either pharmacologic modulator alone could inhibit tumor growth. However, the combination of GCV and modulator produced strong tumor growth delay with some complete regressions. New results demonstrate a novel mechanism for the synergistic bystander killing with HSV-TK/GCV and cytosine deaminase (CD)/5- flucytosine (5-FC), a suicide gene model that produces the anticancer drug 5-fluorouracil. In addition, we demonstrate the importance of sequential drug administration with this double suicide gene therapy for synergistic killing. We will extend these results in murine models of prostate cancer through determining the impact of drug sequencing in preparation for clinical trials. In addition, we propose mechanistic studies designed to elucidate the type and frquency of DMA damage induced by HSV-TK/GCV alone and the impact of modulation with dFdCyd, hydroxyurea or CD/5-FC, as well as the pathways involved in repair of this damage. We will utilize genetic manipulation of human tumor cells as well as a yeast genetic deletion model to identify genes important for DNA damage and repair with these therapeutics. The results will aid us in optimizing current gene therapy protocols as well as initiate novel approaches for greater efficacy.